5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein–Protein Interaction-Derived Functions

A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein–protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure–activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure–activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.


■ INTRODUCTION
−4 These interactions result in increased αSyn and Tau aggregation, decreased PP2A activity and autophagy, and increased reactive oxygen species (ROS) production. 5bnormal processing and aggregation of αSyn and Tau are thought to be the key players in cellular toxicity in Parkinson's disease (PD) and Alzheimer's disease (AD), respectively, 6,7 and decreased PP2A levels and activity are detected in various neurodegenerative diseases (NDDs) such as PD, AD, and other tauopathies. 8,9−19 Originally it was believed that PREP inhibition, i.e., loss of conformational freedom of the enzyme upon inhibitor binding to the proteolytic active site, was responsible for modulating these PPIs.−19 Contrary to KYP-2047, another well-known potent PREP inhibitor S-17092, which is also the most recent PREP inhibitor that entered clinical trials, 20 does not affect multiple PPI-derived functions of PREP in our assays. 17Furthermore, several weak inhibitors are significantly more effective modulators of the PPI-derived functions than KYP-2047.
In our efforts to clarify why PREP inhibition does not correlate with the effects of PPI-derived functions, we challenged the known SAR for PREP ligands.Typical PREP inhibitors are based on a peptide-like scaffold and contain two amide carbonyl groups that are essential for binding to the proteolytic active site (Figure 1). 21Currently, the most structurally distinct PREP inhibitors are our recently published 5-aminooxazoles, which lack both carbonyl groups. 18Although the 5-aminooxazole series contained several potent compounds, both in terms of inhibiting the proteolytic activity of PREP and modulating its PPI-mediated functions, the varying stability of 5-aminooxazoles restricted the full exploration of different amino groups in the 5-position and limited it to (S)-2-cyanopyrrolidine for the biologically active compounds.HUP-55 is the most potent compound in the oxazole series (Figure 1).
In this study the 5-membered heteroaromatic scaffold was further explored by replacing the oxazole ring with a thiazole ring (Figure 1).The 5-aminothiazoles are far more stable than the 5-aminooxazoles for the exploration of different substituents.The compounds were developed solely with a focus on their effect on αSyn dimerization and autophagy, which are the two most studied PPI-derived functions of PREP.We also set out to explain the disconnected SARs using molecular modeling supported by point mutation studies.

■ RESULTS AND DISCUSSION
Synthesis.The thiazoles were initially synthesized via route A of Scheme 1.This route was planned to access the thiazole analogues of HUP-55 with and without the nitrile group, and other analogues could also be synthesized via this route.In route A, 4-methylthiazole 1 was first brominated at the 2 and 5 positions, resulting in compound 2. 22 Compound 2 was then reacted via a Suzuki reaction with trans-3-phenyl-1-propen-1ylboronic acid and trans-2-phenylvinylboronic acid, resulting in compounds 3 and 4, respectively.This reaction was completely selective for bromine in the 2-position.Attempts to directly react the bromine in the 5-position resulted in a mixture of correct product and another product where the bromine had reacted but the amine was attached to the conjugated double bond in the 2-position.We therefore found it necessary to first reduce the alkene.Unfortunately, catalytic hydrogenation with Pd/C resulted in dehalogenation.However, when compounds 3 and 4 were reacted with the milder reducing reagent N,Obis(trifluoroacetyl)hydroxylamine, 23 we obtained compounds 5 and 6, respectively, with a conversion of ca.75% without any dehalogenation.The desired amine could then be introduced via a nucleophilic aromatic substitution reaction on the bromine to the 5-position of the thiazole ring.Finally, the amide of compound 11 was dehydrated to a nitrile using TFAA to obtain compound HUP-46, which was then reacted with NaN 3 to obtain the corresponding tetrazole, compound 13. 16n alternative route was chosen to synthesize a wider series of analogues (route B in Scheme 1).This route was similar to the primary route we used in the synthesis of 5-aminooxazoles. 18The central peptidic intermediates 15a−k were synthesized in one step from suitable amino acid derivatives, such as amino acid pyrrolidides 14a−k or N-(4phenylbutanoyl)glycine 14l.Lawesson's reagent was used to form the thiazole ring and obtain the final 5-aminothiazole products 16a−l. 24Finally, compound 16g was methylated to compound 17.Compounds HUP-46 and 7 were later also synthesized using route B, resulting in better overall yields than that with route A.
In Vitro and Cellular Screening Results of Synthesized Compounds.The thiazoles were tested for their ability (1) to inhibit the proteolytic activity of PREP using recombinant porcine PREP due to its similar structure to human PREP (hPREP) (97% similarity), 27 (2) to block αSyn dimerization in a cellular protein fragment complementation assay (PCA), (3) to induce autophagy in a green fluorescent protein (GFP)-tagged microtubule-associated proteins' 1A/1B light chain 3B (LC3B) expressing human embryonic kidney 293 (HEK-293) cell culture, and (4) to reduce ROS production in FeCl 2 -and H 2 O 2 -stressed SH-SY5Y cells, as previously described for 5-aminooxazoles. 18The results are presented in Table 1.In the autophagy assay, a decreased GFP signal compared to the DMSO control is indicative of increased autophagic flux.Rapamycin, a potent autophagy inducer, reduces the GFP signal to about 65% of the baseline (Supporting Information Figure S51), which can be considered the maximum effect a compound can have in this assay.Compounds that reduce the fluorescence signal in the αSyn dimerization assay and the GFP signal in the autophagy assay below 90% of the baseline can be considered active, as the in vivo effective reference compounds KYP-2047 and HUP-55 18 reduce them to 87 and 89%, respectively.
A comparison of the inhibitory potencies of 5-aminooxazole HUP-55 (5 nM) and the corresponding 5-aminothiazole, HUP-46 (8 μM), supports the postulated binding mode for HUP-55 at the active site, where the oxygen atom in the oxazole ring is involved in a hydrogen bond (Supporting Information Figure S42). 18Replacing the oxazole oxygen with sulfur in thiazoles removes this binding interaction, which could explain the decrease in inhibitory potency.This is a general characteristic for all thiazoles in our series, and none of them are nanomolar inhibitors of the proteolytic activity of PREP.Despite the weak inhibitory potency, HUP-46 was an even more potent modulator of αSyn dimerization and autophagy than HUP-55 or KYP-2047.Compound 7 was equipotent to HUP-46 in modulating αSyn dimerization and only slightly less active in modulating autophagy, although compound 7 lacks the nitrile group.
Compound 11 demonstrated that an amide group cannot be placed in the position of the nitrile group as it had no effect on αSyn dimerization; however, its effect on autophagy was similar to that of compound 7.It should be noted that the purity of this synthesis intermediate was only determined by NMR.Other substituents on the pyrrolidine ring gave inactive compounds.Interestingly, increasing the size of the pyrrolidine ring to a piperidine ring in compound 8 removed the activity on autophagy but only slightly weakened the effect on αSyn dimerization compared to compound 7.
Shortening the linker length in the 2-position of compound 7 by one carbon, resulting in compound 12, removed the effect on αSyn dimerization but maintained the effect on autophagy.Compound 16a, where the phenyl group was 3,4-dimethoxysubstituted, also maintained the effect on autophagy while losing the effect on αSyn dimerization.Other replacements in the 2-position of the thiazole ring typically resulted in a loss of effect in both assays.as the 2-substituent, had an effect on autophagy while lacking an effect on αSyn dimerization.Its N-methylated analogue, compound 17, was inactive in both assays.Replacing the 3indolyl group with a 1-benzimidazolyl group resulted in compound 16h, which showed a comparable effect in both assays to HUP-46 and compound 7.However, the corresponding 2-benzimidazolyl analogue was inactive in both assays.
Replacing the methyl group in the 4-position of compounds 7 and 16g with an isopropyl group, resulting in compounds 16j and 16k, respectively, also led to a loss of activity for both PPImediated functions.This is opposite to what we saw with oxazoles, where a small increase in the size of this substituent improved the activity.It should be noted that in the case of the corresponding oxazoles, the 5-substituent was always (S)-2cyanopyrrolidine.Removing the methyl group in the 4-position resulted in compound 16l, which had an effect on autophagy but not on αSyn dimerization.
Interestingly, several thiazoles were highly effective at reducing the ROS levels in cells with oxidative stress.Etelaïnen et al. 5 showed that KYP-2047 reduces ROS levels by reducing the activity of NADPH oxidase via PP2A activation.Therefore, autophagy and ROS reduction results should have a correlation, but it appears not to be the case with thiazoles.However, thiazoles have been reported to have antioxidant properties themselves, 28 and this may explain the discrepancy between the autophagy induction and ROS production.
HUP-46, 7, and 16h were the most potent modulators of the PPI-mediated functions of PREP in our assays.HUP-46 was the overall most effective compound, being equipotent to compound 7 in reducing αSyn dimerization and equipotent to compound 16h in increasing autophagy, and it was therefore chosen for further testing in the cellular assays.Overall, HUP-46 is one of the most potent modulators of the PPI-mediated functions of PREP that has so far been reported.
Biological Characterization of HUP-46.After the cellular screening assays, we wanted to assess the concentration−response effect of HUP-46 on αSyn dimerization and autophagic flux in GFP-LC3B-RFP HEK-293 cells.In the αSyn  18 The ratio was significantly decreased to 1 μM concentration.A dose-dependent effect was seen on LC3BII levels in HEK-293 cells after 4 h incubation (E).An autophagic flux assay showed significantly elevated LC3BII levels with 20 nM bafilomycin A1 and 10 μM HUP-46 after 4 h incubation (F), confirming the autophagic flux activation.A significant effect on pPP2A was also seen in mouse primary neurons (G).In an αSyn PCA with 10 μM lactacystin (LACTA) (10 μM) as a positive control, no effect with 10 μM HUP-46 was seen on αSyn dimerization in PREP-KO cells (H).Similarly, no effect with 10 μM HUP-46 and 20 nM bafilomycin was seen on LC3BII levels in PREP-KO HEK-293 cells (I).Data are presented as mean ± SEM *, p < 0.05, **, p < 0.01; ***, p < 0.001; one-way ANOVA with Tukey's posthoc test (A−D,G,H) and two-way ANOVA with Sidak's posthoc test (F,I); *, #, p < 0.05, two-way ANOVA with Sidak's posthoc test (F).dimerization assay, the maximal efficacy was achieved at 10 μM concentration, and, similar to HUP-55, 18 higher concentrations did not show an additional effect (Figure 2A; p < 0.01 10 μM; p < 0.05 1 μM HUP-46 compared to DMSO, one-way ANOVA with Tukey's post hoc test).In the autophagic flux assay, the effect was concentration-dependent up to 20 μM, but 50 μM restored the autophagic flux to control levels (Figure 2B).This might be indicative for toxicity-induced changes in autophagic flux, as 100 μM concentration showed toxicity in neuronal and non-neuronal cells (Supporting Information Figure S52).Moreover, the restored autophagic flux may also contribute to the elevated signal in αSyn dimerization assay with 50 μM concentration (Figure 2A).PP2A activation and autophagy were then measured in nonreporter cells.A 4 h incubation in HEK-293 cells increased the levels of the total catalytic subunit of PP2A (PP2Ac; Figure 2C,D) and significantly decreased the levels of inactive PP2Ac (pPP2Ac; specificity for inactive PP2A tested by Svarcbahs et al. 3  Based on the pPP2A/PP2A ratio, an EC 50 value of 100 nM was calculated for HUP-46 (Figure 2C), whereas for HUP-55 from the oxazole series, the EC 50 value was 275 nM. 18In addition to potency, maximal efficacy on the pPP2A/ PP2A ratio was better for HUP-46 than for HUP-55 (Figure 2C; 0.18 vs 0.32).
HUP-46 was also tested in mouse primary neurons, where it significantly decreased pPP2A levels with both 1 and 10 μM concentrations (Figure 2G; p < 0.01 compared to NC, oneway ANOVA with Tukey's post-test).The impact of HUP-46 on αSyn dimerization and autophagy is PREP-specific as there was no effect on αSyn dimerization (Figure 2H) or autophagic As these compounds are being developed to target PREP in the brain, HUP-46 was also shown to penetrate the blood− brain barrier by measuring its brain concentration in mice after i.p. injection (Supporting Information Figure S61).
Identifying a Possible Binding Site Using Molecular Modeling.So far, we have been unable to give an explanation for the disconnected SARs for the inhibition of the proteolytic activity and modulation of the PPI-mediated effects of PREP.A previous study using molecular dynamics (MD) simulations identified some subtle differences in PREP dynamics when three different ligands were bound to the active site, and the authors hypothesized that this could be the reason behind the differences in their ability to modulate the PPI between αSyn and PREP. 29Although the ligands chosen for that study differ in their efficacy in modulating αSyn aggregation and autophagy, 17 they are all potent peptide-like inhibitors of PREP, and this hypothesis does not explain how very weak inhibitors can modulate the PPI-mediated functions.
Our hypothesis was that the most likely explanation is the presence of another binding site, separate enough from the active site so that ligand binding to this other site does not prevent substrate binding.Additionally, we hypothesized that there should be similarities in ligand binding at the other binding site and the catalytic binding site.This would allow ligands like KYP-2047, which in addition to being modulators of the PPI-mediated functions are potent inhibitors, to bind to both the active site and this other site, with their affinity to each site determining their activity profile.
Molecular modeling was used to identify possible binding sites.These studies were performed with the only available crystal structure of hPREP (PDB ID: 3DDU). 30Using Maestro's SiteMap tool, 31 two sites inside the cavity of PREP, near the hinges connecting the two subunits, were identified as potential new binding sites (orange and purple in Figure 3A).They both had a SiteScore of 1.0, which is equal to that of the active site.The 5-aminothiazoles in Table 1, the previously published 5-aminooxazoles, 18 and selected peptidelike ligands 16,19 were docked to the two binding sites using two methods: Glide and Induced Fit. 31 Based on the SiteScores and visual inspection of the binding poses, the hinge site located on the catalytic subunit (purple in Figure 3A) was chosen for further inspection.Induced fit docking results to this site for selected compounds are shown in Figures 3 and  S22.The distance between this potential new binding site and the active site is about 15−20 Å, as shown in Figure 3C where HUP-55 is docked to both sites.Interestingly, Tyr473 at the active site, which is important for the proteolytic activity of PREP, and Tyr471 at the other binding site, which seems to be important especially for thiazole and oxazole binding, are separated by just one glycine residue.
The binding interactions and viability of the binding site were further assessed with MD simulations using Desmond. 31D simulations were run for HUP-46, HUP-55, and HUP-28.

Journal of Medicinal Chemistry
also chosen for point mutation to cysteine.The rationale for the cysteines was to have the option to attach covalently binding analogues of HUP-28 and compound 7, without significantly altering the predicted binding poses of the parent compounds (Supporting Information Figure S50).The residues chosen for point mutations are highlighted in Figure 3B,C.
Mutations at the New Binding Pocket Do Not Affect Proteolytic Activity.Based on molecular modeling and preliminary biological characterization, we prepared PREP constructs with the following point mutations at the putative new binding pocket: Tyr471Ala, Asn483Ala, Ser485Ala, and Leu499Cys.PREP-KO HEK-293 cells were transfected with hPREP and the mutant constructs, and their enzymatic activity was assessed with a fluorogenic substrate-based assay (Suc-Gly-Pro-AMC) and with activity-based protein profiling (ABPP).The results show that the active site functions remain mostly unchanged in PREP-KO HEK-293 cells transfected with different PREP mutants in the fluorogenic assay (Figure 4A; PREP protein levels after transfection and full ABPP membrane with loading control are shown in Supporting Information Figure S54) and in the ABPP assay (Figure 4B).Interestingly, Tyr471Ala PREP showed elevated PREP activity in the substrate-based assay (Figure 4A; p < 0.001 compared to hPREP, one-way ANOVA with Tukey's posthoc test) but decreased signal in the ABPP assay (Figure 4B,C; p < 0.001 compared to hPREP, one-way ANOVA with Tukey's posthoc test).Tyr471 is the closest point to the active site, and mutations in that residue may also change the proteolytic activity.On the other hand, the ABPP assay shows the availability of the active serine (Ser554) for the ABPP probe, and this may be different than the substrate−cleavage activity.
Additionally, the sensitivity of PREP mutants to HUP-46 and peptidic PREP ligands KYP-2091 32 and KYP-2112 33 was tested using mutant recombinant hPREP (Asn483Ala and Leu499Cys) in a fluorogenic assay (Figure 4E,F).KYP-2112 is a potent PREP inhibitor (IC 50 0.32 nM) 33 with no PPI-related effects, and KYP-2091 is a weak inhibitor (IC 50 1 μM) 32 with moderate PPI effects. 17When measuring IC 50 using the fluorogenic Suc-Gly-Pro-AMC substrate in hPREP and the Asn483Ala and Leu499Cys mutants, no clear differences between hPREP and mutants were seen in any of the compounds (Figure 4D−F).Moreover, the baseline activity between mutants and hPREP was not changed in the assay.In the ABPP assay, the ability of a compound to prevent the ABPP probe from binding to the active serine of PREP was tested in hPREP and the mutants.The results obtained with each of the test compounds did not significantly differ between hPREP and the mutants (Supporting Information Figure S55A−C).Taken together, it appears that mutations in the new binding site do not have any effect on the proteolytic activity of PREP.
HUP-46 Favors the New Binding Pocket in a Cellular Thermal Shift Assay.To characterize the binding of different ligands to the new binding pocket, we performed a cellular thermal shift assay (CETSA) with PREP-KO HEK-293 cells that were transfected with hPREP or PREP mutant constructs.KYP-2091 and KYP-2112 were used as the reference compounds for HUP-46.
CETSA analysis showed that all compounds bind to hPREP (Figure 5A−C), and the aggregation temperatures (T agg ) are presented in Table 2. Mutations of Asn483Ala, Leu499Cys, and Tyr471Ala completely abolished the binding of HUP-46 5D,G,J; Table 2) but had no effect on the binding of KYP-2112 (Figure 5F,I,L; Table 2).Compared to KYP-2112, KYP-2091 showed reduced binding to the Leu499Cys and Asn483Ala mutants and no binding to the Tyr471Ala mutant in this cell-based assay (Figure 5E,H,K).CETSA results for Ser485Ala were nonconclusive, and they were left out of this analysis.The reduced binding of KYP-2091 to the Asn483Ala and Leu499Cys mutants was verified using an isothermal titration calorimetry (ITC) assay, indicating that its binding constant (K d ) was increased fourfold in both mutants.The Leu499Cys mutation had no effect on the binding of KYP-2112, and the Asn483Ala mutation even improved the K d value of KYP-2112 (Table 3; see Supporting Information Tables S2  and S3 and Figures S56−S59 for further details).
When comparing their biological efficacy in PPI-related functions, there was a clear correlation between binding to the new binding pocket and PP2A activation when determined by the pPP2A/PP2A ratio (Figure 6A,B).KYP-2112 did not decrease the pPP2A/PP2A ratio, and KYP-2091 had a maximal effect of 0.55 (Figure 6A).Additionally, HUP-46 did not have an effect on PP2A activation in PREP-KO HEK-293 cells expressing Asn483Ala, Leu499Cys, Tyr471Ala, or Ser485Ala PREP and showed efficacy in pPP2A only in hPREPtransfected cells (Figure 6C,D).Similar results were seen in LC3BII levels, where HUP-46 had an impact on hPREPtransfected cells but not in mutant-transfected cells (Supporting Information Figure S60).

■ CONCLUSIONS
The novel 5-aminothiazoles are more potent and effective modulators of the PPI-derived functions of PREP and are advantageous over the previously reported 5-aminooxazoles as they are weaker inhibitors of the proteolytic activity of PREP, and as evidence shows, strong inhibition of the proteolytic activity is not required for the effective modulation of the PPImediated functions.Furthermore, 5-aminothiazoles are generally more stable than 5-aminooxazoles, allowing a wider variation of different substituents.The novel 5-aminothiazoles HUP-46, 7, and 16h are among the most effective modulators of the PPI-related functions of PREP reported to date,  although they have only weak, micromolar inhibitory potency.
Although HUP-46 is the most effective of these three in biological screening assays, the other two clearly demonstrate that the electrophilic nitrile group and the lipophilic phenylpropyl group can be replaced in the structure.HUP-46 was subjected to further cellular characterization, where it significantly reduced the ratio of pPP2A/PP2A, increased the autophagy marker LC3BII levels, and decreased αSyn dimerization in a concentration−response manner.The effect was confirmed to be PREP-specific by using PREP-KO cells.Furthermore, it should be highlighted that in addition to HUP-46 being a more effective modulator of PP2A activity compared to the corresponding oxazole HUP-55, it is also a more potent modulator of PP2A activity.As HUP-46 is also a brain-penetrating compound, it is a useful molecular probe for the development of therapeutic compounds for neurodegenerative diseases.
Our hypothesis explaining the disconnected SARs was that the PPI-mediated functions of PREP are not modulated through the proteolytic active site.Using molecular modeling, we identified a potential new binding site inside the cavity of PREP, which was clearly separate from the proteolytic active site.MD simulations were used to examine the most important residues for binding to this site, and based thereon, PREP constructs with different point mutations at the postulated new binding site were prepared.Mutations at the new binding site did not have significant effects on the proteolytic activity of PREP or on the inhibitory potency of the compounds.The effect of point mutations on ligand binding was determined using CETSA and ITC.HUP-46 and KYP-2091, both weak inhibitors of PREP but effective modulators of PPI-mediated functions, were more affected by the mutations than KYP-2112, a potent inhibitor of PREP with no activity on the PPImediated functions.Additionally, unlike in cells expressing wild-type PREP, HUP-46 was unable to affect PP2A, pPP2A, or LC3BII levels in cells expressing PREP with the different point mutations.We therefore conclude that there is another ligand-binding site inside the cavity of PREP, located around the described point mutations, which is more important for the modulation of the PPI-mediated functions of PREP than the proteolytic active site.
■ EXPERIMENTAL SECTION Chemistry.General Information.Unless otherwise specified, all reagents and solvents were obtained from commercial suppliers and used without purification.Microwave reactions were performed with a fixed hold time in capped microwave vials using a Biotage Initiator+ (Biotage).Completion of reactions and purifications were monitored with TLC, which was performed on 60 F 254 silica gel plates, using UV light (254 and 366 nm) and ninhydrin or iodine staining to detect products.Flash chromatography was performed manually with silica gel (230−400 μm mesh) or using a Biotage Isolera One (Biotage) with silica gel 60 (40−63 μm mesh), unless otherwise specified. 1H and 13 C NMR spectra were recorded at 400 and 101 MHz, respectively, using an Ascend 400 (Bruker).CDCl 3 was used as the NMR solvent unless otherwise specified.Chemical shifts (δ) are reported in parts per million (ppm) with TMS or solvent residual peaks as reference.Exact mass and purity of the tested compounds were analyzed with LC−MS, using a Waters Aquity UPLC system (Waters) and a Waters Synapt G2 HDMS mass spectrometer (Waters) via an ESI ion source in positive mode.The synthesis of peptidic starting materials 15a−l is reported in the Supporting Information.The purity of all tested final compounds was 95% or higher, except compound 8, which had a purity of 94%.
PREP Activity Assay.To determine IC 50 values against PREP, we used purified recombinant porcine PREP.PREP enzyme was purified according to the protocol described by Venalaïnen et al. 26 In the microplate assay procedure, 10 μL of the enzyme dilution was preincubated with 65 μL of 0.1 M sodium−potassium phosphate buffer (pH 7.0) containing the compounds at different concentrations at 30 °C for 30 min.The reaction was initiated by adding 25 μL of 4 mM Suc-Gly-Pro7-amido-4-methylcoumarin dissolved in 0.1 M sodium−potassium phosphate buffer (pH 7.0), and the mixture was incubated at 30 °C for 60 min.The reaction was terminated by adding 100 μL of 1 M sodium acetate buffer (pH 4.2).Formation of 7amido-4-methylcoumarin was determined fluorometrically with a microplate fluorescence reader (excitation at 360 nm and emission at 460 nm).The final concentration of the compounds in the assay mixture varied from 100 μM to 1 nM, and the final concentration of the enzyme was approximately 2 nM.The inhibitory activities (percent of control) were plotted against the log concentration of the compound, and the IC 50 value was determined by nonlinear regression utilizing GraphPad Prism 3.0 software.
In cell cultures, the PREP activity was measured, as in the study of Myoḧanen et al. 11 The cells were homogenized with lysis buffer (50 mM KH 2 PO 4 , 1.5 mM MgCl 2 , 10 mM NaCl, 1 mM EDTA; pH 7.4).Cell homogenates were centrifuged at 16,000g for 10 min at +4 °C.The PREP activity was measured from supernatants using Suc-Gly-Pro7-amido-4-methylcoumarin substrate as above.The protein concentration was measured by BCA, and the specific activity was correlated to the protein amount.
α-Synuclein Dimerization Assay.To study the effect of compounds on the early phases of αSyn aggregation, αSyn dimerization was assessed by using a protein fragment complementation assay (PCA) that is slightly modified from that by Savolainen et al. 2 and used by us (Kilpelaïnen et al. 18 and in Paẗsi et al). 19Briefly, N2A cells were seeded on 96-well plates (Isoplate white wall, PerkinElmer Life Sciences) at the density of 13,000 cells/well and transfected with 25 ng of both αSyn-Gluc1 and αSyn-Gluc2 or 50 ng mock-plasmid as a control by using Lipofectamine 3000 (L3000001; Thermo Fischer Scientific) as the transfection reagent; 48 h posttransfection, cells were incubated for 4 h with the study compounds (10 μM).0.1% DMSO served as the vehicle control, and proteasomal inhibitor lactacystin (L-1147; AG Scientific, San Diego, CA) at 10 μM served as a positive control for αSyn dimerization.The PCA signal was assessed by injecting 25 μL of native coelenterazine (Nanolight Technology) in phenol red-free DMEM per well.The emitted luminescence was read using a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific).For each experimental condition, four replicate wells were used, and at least three separate experiments for each treatment.
Autophagic Flux Assay.To assess the effect of compounds on autophagy, autophagic flux was determined by using HEK-293 cells with stable GFP-LC3B-RFP construct expression.The assay was performed as described by Svarcbahs et al. 3 and by Kilpelaïnen et al. 17 Briefly, the cells were seeded at a density of 30,000 cells/well on black, clear-bottomed 96-well plates (Costar, Corning) and treated for 24 h with the study compounds 24 h postplating (10 μM; 0.1% DMSO as the vehicle control).0.5 μM rapamycin, an mTOR inhibitor (BML-A275; Enzo Life Sciences), was used as a positive control for autophagy induction and 20 nM bafilomycin 1A (ML1661) as an autophagy inhibitor.Twenty-four hours after treatment, cells were washed once with warm PBS, and GFP signal was read with Victor2 multilabel counter (PerkinElmer; excitation/ emission 485 nm/535 nm).For each experimental condition, four replicate wells were used in each experiment, and at least three independent experiments were performed.
ROS Detection Assay.The impact of compounds on ROS production under oxidative stress (OS) was assessed as we have done earlier in the study of Etelaïnen et al. 5 In short, SH-SY5Y cells were plated on a clear bottom black-walled 96-well plate (30,000 cells per well) and incubated overnight.OS was induced treating the cells with a culture medium including 100 μM H 2 O 2 (H1009; Merck) and 10 mM FeCl 2 (44939-50G) with or without concurrent treatment compounds for 3 h (10 μM).The cells in the control wells received only fresh cell growth medium during OS induction.Stress-induced ROS production was studied using the DCFDA cellular ROS detection assay kit (ab113851, Abcam) according to the protocol provided with it.ROS proportional fluorescence signal was measured with Victor2 multilabel counter (PerkinElmer; excitation/emission 485 nm/535 nm).
The following day, the membranes were washed, followed by a 2 h incubation at room temperature with Gt-anti-Rb (#31460, Invitrogen, 1:2000).After incubation, the membranes were washed and incubated with SuperSignal West Pico (#34577) or Femto (#34095) Chemiluminescent Substrate (Thermo Fisher Scientific) for 5 min, and the images were captured with the ChemiDoc XRS+ Gel Imaging System (Bio-Rad) controlled by ImageLab software (version 6.01, Bio-Rad).
To verify that bands were in the linear range of detection, increasing exposure time and automatic detection of saturated pixels in ImageLab software (version 6.01, Bio-Rad) were used.Thereafter, images were converted to 8-bit grayscale format, and the OD (arbitrary units, a.u.) of the bands was measured with ImageJ (histogram area analysis; version 1.53c; NIH).The OD obtained from each band was normalized against the corresponding vinculin band, which was used as the loading control.
Cellular Thermal Shift Assay.After the 24 h transfection with PREP mutants (Asn483Ala, Leu499Cys, Tyr470Ala, and Ser485Ala), the cells were exposed to HUP-46, KYP-2091, or KYP-2112 (10 μM) in the medium for 2 h.After the exposure, the cells were collected in PBS and aliquoted into seven PCR tubes (100,000 cells/tube).The cells were prewarmed at 37 °C for 3 min, then heated to 37, 47, 50, 53, 56, 63, or 67 °C for 3 min, and subsequently cooled at 25 °C for 3 min using a PCR Mastercycler (T100 Thermal Cycler, Bio-Rad).After the heating, the cells were disrupted with two freeze−thaw cycles by submerging the tubes into liquid nitrogen and subsequently thawed by incubation at 25 °C for 3 min.The aggregated proteins were removed by centrifugation (at 20,000g for 20 min at 4 °C), and the soluble fractions were diluted with Laemmli buffer (Bio-Rad, Hercules, CA, USA) and analyzed by Western blot as described above.The nondenaturated protein fractions (%) were calculated by comparing the intensities of temperature-treated cell samples to the corresponding cell samples from 37 °C.

■ ASSOCIATED CONTENT
* sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.

Figure 1 .
Figure 1.Peptide-like PREP inhibitors KYP-2047 and HUP-28, both of which are also strong modulators of the PPI-mediated functions of PREP.S-17092 is a peptide-like potent PREP inhibitor lacking an effect on multiple PPI-mediated functions of PREP.The structurally distinct nonpeptidic 5-aminooxazole-based PREP inhibitor HUP-55 and its new 5-aminothiazole-based analogue HUP-46 presented in this paper.

Figure 3 .
Figure 3. (A) Ribbon representation of the crystal structure of hPREP (PDB ID: 3DDU) 30 , with 5-aminooxazole HUP-55 docked into the active site (green) and two other possible binding sites (purple and orange).(B) Induced fit docking pose for HUP-46 at the postulated new binding site.(C) Induced fit docking poses of HUP-55 at both the active site and the postulated new binding site, with the measured distance between the oxazole rings of 18.62 Å.The amino acid residues chosen for mutation studies are colored orange.Hydrogen bonds are shown as yellow dashed lines and π−π stacking as blue dashed lines.

Figure 4 .
Figure 4. Characterization of PREP with mutations in the novel binding pocket.Tyr471Ala PREP showed increased activity when transfected to PREP knockout HEK-293 cells in the fluorogenic substrate-based activity assay (A) but had reduced activity in activity-based protein profiling assay [ABPP; (B)].Representative bands from the ABPP assay (C).The inhibition of PREP proteolytic activity by HUP-46, KYP-2091, and KYP-2112 was tested in recombinant hPREP and Asn483Ala and Leu499Cys PREP mutants by using the fluorogenic substrate-based assay (D−F).***, p < 0.001, one-way ANOVA with Tukey's posthoc test.

Table 1 .
19ological Activities of the Synthesized 5-Aminothiazoles i Assessed using recombinant porcine PREP with Suc-Gly-Pro-AMC as the substrate.bLuminescencesignalpercentage of DMSO control with SEM, assessed with a split Gaussia luciferase-based method using Neuro2A cells.cGFPsignalpercentage of DMSO control with SEM, assessed using HEK-293 cells stably expressing GFP-LC3B.dFluorescencesignalpercentage of DMSO control with SEM, assessed using a fluorogenic ROS assay.eResultsreportedbyKilpelaïnenetal.18 fThe assay is limited by the enzyme concentration of 2 nM for IC 50 values under this concentration; KYP-2047 is a slow, tight-binding inhibitor with a K i -value of 0.02 nM.25,26gResultsexceptROSassay reported by Kilpelaïnen et al.16or Paẗsi et al.19h Synthesis intermediate.i n.d.: not determined. a

Table 3 .
K d Values for Binding to PREP and Two Mutants for KYP-2112 and KYP-2091, Determined Using ITC a a KYP-2091 has a higher K d value when binding to mutated PREP compared to wild-type PREP.Similar effect was not observed with KYP-2112.*p < 0.05.**p < 0.01; Student's t test.